Poly I:C elicits broader and stronger humoral and cellular responses to a Plasmodium vivax circumsporozoite protein malaria vaccine than Alhydrogel in mice.

Malaria remains a global health challenge, necessitating the development of effective vaccines. The RTS,S vaccination prevents Plasmodium falciparum (Pf) malaria but is ineffective against Plasmodium vivax (Pv) disease. Herein, we evaluated the murine immunogenicity of a recombinant PvCSP incorporating prevalent polymorphisms, adjuvanted with Alhydrogel or Poly I:C. Both formulations induced prolonged IgG responses, with IgG1 dominance by the Alhydrogel group and high titers of all IgG isotypes by the Poly I:C counterpart. Poly I:C-adjuvanted vaccination increased splenic plasma cells, terminally-differentiated memory cells (MBCs), and precursors relative to the Alhydrogel-combined immunization. Splenic B-cells from Poly I:C-vaccinated mice revealed an antibody-secreting cell- and MBC-differentiating gene expression profile. Biological processes such as antibody folding and secretion were highlighted by the Poly I:C-adjuvanted vaccination. These findings underscore the potential of Poly I:C to strengthen immune responses against Pv malaria.

In Vitro Generation of Human Antibody-Secreting Cells Through the Stimulation of PBMCs with Dengue Virus Particles.

The Dengue pathophysiology has had several aspects determined over the years. However, some points remain elusive, such as the metabolic factors that regulate the massive B cell differentiation into antibody-secreting cells observed in Dengue patients. In this chapter, we describe an in vitro method capable of mimicking this Dengue-induced cell expansion. More specifically, this approach allows dengue virus-stimulated peripheral blood mononuclear cells (PBMCs) from healthy individuals to enhance the frequency of phenotypical and functional antibody-secreting cells (ASCs) after 7 days of culture. A manuscript recently published by Bonezi and colleagues displays results generated through this methodology.

Viremia controls Env-specific antibody-secreting cell responses in simian immunodeficiency virus infected macaques pre and post-antiretroviral therapy.

OBJECTIVE: The aim of this study was to investigate the kinetics of Env (gp140)-specific antibody-secreting cells (ASCs) during acute and early chronic simian immunodeficiency virus (SIV) infection, and prior to and postantiretroviral therapy (ART) in rhesus macaques. DESIGN AND METHODS: At week 0, rhesus macaques were inoculated intravenously with SIVmac239 and the viral loads were allowed to develop. Daily ART was initiated at week 5 post infection until week 18, though the animals were monitored until week 28 for the following parameters: enumeration of SIV gp140-specific ASCs by ELISPOT; quantification of viremia and SIV gp140-specific IgG titres through qRT-PCR and ELISA, respectively; estimation of monocytes, follicular helper T cells (Tfh) and memory B cell frequencies using polychromatic flow cytometry. RESULTS: Direct correlations were consistently found between blood SIV gp140-specific ASC responses and viremia or SIV Env-specific IgG titres. In contrast, SIV gp140-specific ASC responses showed inverse correlations with the percentage of total memory B cells in the blood. In lymph nodes, the magnitude of the SIV gp140-specific ASC responses also followed the viral load kinetics. In contrast, the number of SIV gp140-specific ASCs presented did not correlate with frequencies of circulating activated monocyte (CD14+CD16+) or Tfh cells. CONCLUSION: Blood and/or lymph node viral loads may regulate the onset and magnitude of SIV gp140-specific ASCs during SIV infection and following ART in rhesus macaques.

Antibodies Against the Plasmodium vivax Apical Membrane Antigen 1 From the Belem Strain Share Common Epitopes Among Other Worldwide Variants.

Malaria is a human parasitic disease distributed in many tropical countries and caused by various Plasmodium species. Plasmodium vivax has the largest geographical distribution of the Plasmodium species and is predominant in the Americas, including Brazil. Only a small number of P. vivax vaccine formulations have successfully reached clinical trials relative to their P. falciparum counterparts. One of the candidate antigens for a blood-stage P. vivax vaccine is apical membrane antigen 1 (PvAMA-1). Due to the worldwide distribution of Plasmodium parasites, a high degree of variability has been detected in this antigen sequence, representing a considerable challenge to the development of a universal vaccine against malaria. In this study, we evaluated how PvAMA-1 polymorphisms influence vaccine-derived immune responses in P. vivax malaria. To this end, we expressed 9 recombinant protein representatives of different PvAMA-1 allelic variants in the yeast Pichia pastoris: Belem, Chesson I, Sal-1, Indonesia XIX, SK0814, TC103, PNG_05_ESP, PNG_62_MU, and PNG_68_MAS. After protein expression and purification, we evaluated the breadth of the immune responses derived from malaria-exposed individuals from the Amazon region. From 611 serum samples of malaria-exposed individuals, 53.68% of them reacted against the PvAMA-1 Belem through ELISA. Positive samples were further tested against recombinant proteins representing the other PvAMA-1 allelic variants. Whereas Sal-1, Chesson I and SK0814 variants were highly recognized by tested serum samples, Indonesia XIX, TC103, PNG_05_ESP, PNG_62_MU, and PNG_68_MAS were only slightly recognized. Moreover, polyclonal sera derived from C57BL/6 mice immunized with the PvAMA-1 Belem protein predominantly recognized Belem, Sal-1, Chesson I, SK0814, and Indonesia XIX through ELISA. Last, ELISA-based competition assays demonstrated that a previous interaction between anti-Belem polyclonal serum and Sal-1, Chesson I, SK0814, or Indonesia XIX proteins could further inhibit antibody binding to the Belem variant. Our human and mouse data suggest the presence of common epitopes or cross-reactivity between Belem, Sal-1, Chesson I, and SK0814 variants. Although the PvAMA-1 Belem variant induces strain-transcendent antibodies, PvAMA-1 variants from Thailand and Papua New Guinea may need to be included in a universal vaccine formulation to achieve protection against P. vivax malaria.

São Paulo School of Advanced Sciences on Vaccines: an overview.

Two years ago, we held an exciting event entitled the São Paulo School of Advanced Sciences on Vaccines (SPSASV). Sixty-eight Ph.D. students, postdoctoral fellows and independent researchers from 37 different countries met at the Mendes Plaza Hotel located in the city of Santos, SP - Brazil to discuss the challenges and the new frontiers of vaccinology. The SPSASV provided a critical and comprehensive view of vaccine research from basics to the current state-of-the-art techniques performed worldwide. For 10 days, we discussed all the aspects of vaccine development in 36 lectures, 53 oral presentations and 2 poster sessions. At the end of the course, participants were further encouraged to present a model of a grant proposal related to vaccine development against individual pathogens. Among the targeted pathogens were viruses (Chikungunya, HIV, RSV, and Influenza), bacteria (Mycobacterium tuberculosis and Streptococcus pyogenes), parasites (Plasmodium falciparum or Plasmodium vivax), and the worm Strongyloides stercoralis. This report highlights some of the knowledge shared at the SPSASV.

Protective Malaria Vaccine in Mice Based on the Plasmodium vivax Circumsporozoite Protein Fused with the Mumps Nucleocapsid Protein.

Plasmodium vivax is the most common species of human malaria parasite found outside Africa, with high endemicity in Asia, Central and South America, and Oceania. Although Plasmodium falciparum causes the majority of deaths, P. vivax can lead to severe malaria and result in significant morbidity and mortality. The development of a protective vaccine will be a major step toward malaria elimination. Recently, a formulation containing the three allelic variants of the P. vivax circumsporozoite protein (PvCSP-All epitopes) showed partial protection in mice after a challenge with the hybrid Plasmodium berghei (Pb) sporozoite, in which the PbCSP central repeats were replaced by the VK210 PvCSP repeats (Pb/Pv sporozoite). In the present study, the chimeric PvCSP allelic variants (VK210, VK247, and P. vivax-like) were fused with the mumps virus nucleocapsid protein in the absence (NLP-CSPR) or presence of the conserved C-terminal (CT) domain of PvCSP (NLP-CSPCT). To elicit stronger humoral and cellular responses, Pichia pastoris yeast was used to assemble them as nucleocapsid-like particles (NLPs). Mice were immunized with each recombinant protein adjuvanted with Poly (I:C) and presented a high frequency of antigen-specific antibody-secreting cells (ASCs) on days 5 and 30, respectively, in the spleen and bone marrow. Moreover, high IgG titers against all PvCSP variants were detected in the sera. Later, these immunized mice with NLP-CSPCT were challenged with Pb/Pv sporozoites. Sterile protection was observed in 30% of the challenged mice. Therefore, this vaccine formulation use has the potential to be a good candidate for the development of a universal vaccine against P. vivax malaria.

Flavivirus-Mediating B Cell Differentiation Into Antibody-Secreting Cells in Humans Is Associated With the Activation of the Tryptophan Metabolism.

Patients infected with the Dengue virus (DENV) often present with a massive generation of DENV-specific antibody-secreting cells (ASCs) in the blood. In some cases, these ASCs represent more than 50% of the circulating B cells, a higher magnitude than those induced by other infections, vaccinations, and plasma cell lymphomas. However, it remains unclear how the DENV infection elicits this colossal response. To address this issue, we utilised an in vitro strategy to induce human PBMCs of healthy individuals incubated with DENV particles (DENV4 TVP/360) to differentiate into ASCs. As controls, PBMCs were incubated with a mitogen cocktail or supernatants of uninfected C6/36 cells (mock). The ASC phenotype and function were increasingly detected in the DENV and mitogen-cultured PBMCs as compared to mock-treated cells. In contrast to the in vivo condition, secreted IgG derived from the PBMC-DENV culture was not DENV-specific. Lower ASC numbers were observed when inactivated viral particles or purified B cells were added to the cultures. The physical contact was essential between B cells and the remaining PBMCs for the DENV-mediated ASC response. Considering the evidence for the activation of the tryptophan metabolism detected in the serum of Dengue patients, we assessed its relevance in the DENV-mediated ASC differentiation. For this, tryptophan and its respective metabolites were quantified in the supernatants of cell cultures through mass spectrophotometry. Tryptophan depletion and kynurenine accumulation were found in the supernatants of PBMC-DENV cultures, which presented enhanced detection of indoleamine 2,3-dioxygenase 1 and 2 transcripts as compared to controls. In PBMC-DENV cultures, tryptophan and kynurenine levels strongly correlated to the respective ASC numbers, while the kynurenine levels were directly proportional to the secreted IgG titers. Contrastingly, PBMCs incubated with Zika or attenuated Yellow Fever viruses showed no correlation between their kynurenine concentrations and ASC numbers. Therefore, our data revealed the existence of distinct pathways for the DENV-mediated ASC differentiation and suggest the involvement of the tryptophan metabolism in this cellular process triggered by flavivirus infections.

Hydroquinone exposure alters the morphology of lymphoid organs in vaccinated C57Bl/6 mice.

The influenza is a common viral infection that can be fatal, especially in high-risk groups such as children, pregnant women, elderly, and immune-deficient individuals. Vaccination is the most efficient approach to prevent the spreading of viral infection and promote individual and public health. In contrast, exposure to environmental pollutants such as cigarette smoke reduces the efficacy of vaccination. We investigated whether chronic exposure to hydroquinone (HQ), the most abundant compound of the tobacco particulate phase, could impair the adaptive immune responses elicited by influenza vaccination. For this, adult male C57BL/6 mice were daily exposed to either nebulized HQ or PBS for 1 h for a total of eight weeks. At weeks 6 and 8, the mice were primed and boosted with the trivalent influenza vaccine via IM respectively. Although the HQ exposure did not alter the body weight of the mice and the biochemical and hematological parameters, the pollutant increased the oxidative stress in splenocytes of immunized animals, modified the morphology of spleen follicles, and augmented the size of their lymph nodes. The lymphoid organs of HQ-exposed mice presented a similar number of vaccine-specific IgG-secreting cells, titers of vaccine-specific total IgG, and respective subclasses. Transcriptome studies with HQ, benzene, or cigarette smoke exposure were also analyzed. The genes up-regulated upon pollutant exposure were associated with neutrophil migration and were shown to be co-expressed with antibody-secreting cell genes. Therefore, these findings suggest that HQ exposure may trigger an immune-compensatory mechanism that enhances the humoral responses induced by influenza vaccination.

Hydroquinone exposure alters the morphology of lymphoid organs in vaccinated C57Bl/6 mice

The influenza is a common viral infection that can be fatal, especially in high-risk groups such as children, pregnant women, elderly, and immune-deficient individuals. Vaccination is the most efficient approach to prevent the spreading of viral infection and promote individual and public health. In contrast, exposure to environmental pollutants such as cigarette smoke reduces the efficacy of vaccination. We investigated whether chronic exposure to hydroquinone (HQ), the most abundant compound of the tobacco particulate phase, could impair the adaptive immune responses elicited by influenza vaccination. For this, adult male C57BL/6 mice were daily exposed to either nebulized HQ or PBS for 1 h for a total of eight weeks. At weeks 6 and 8, the mice were primed and boosted with the trivalent influenza vaccine via IM respectively. Although the HQ exposure did not alter the body weight of the mice and the biochemical and hematological parameters, the pollutant increased the oxidative stress in splenocytes of immunized animals, modified the morphology of spleen follicles, and augmented the size of their lymph nodes. The lymphoid organs of HQ-exposed mice presented a similar number of vaccine-specific IgG-secreting cells, titers of vaccine-specific total IgG, and respective subclasses. Transcriptome studies with HQ, benzene, or cigarette smoke exposure were also analyzed. The genes up-regulated upon pollutant exposure were associated with neutrophil migration and were shown to be co-expressed with antibody-secreting cell genes. Therefore, these findings suggest that HQ exposure may trigger an immune-compensatory mechanism that enhances the humoral responses induced by influenza vaccination.

Hydroquinone exposure alters the morphology of lymphoid organs in vaccinated C57Bl/6 mice

The influenza is a common viral infection that can be fatal, especially in high-risk groups such as children, pregnant women, elderly, and immune-deficient individuals. Vaccination is the most efficient approach to prevent the spreading of viral infection and promote individual and public health. In contrast, exposure to environmental pollutants such as cigarette smoke reduces the efficacy of vaccination. We investigated whether chronic exposure to hydroquinone (HQ), the most abundant compound of the tobacco particulate phase, could impair the adaptive immune responses elicited by influenza vaccination. For this, adult male C57BL/6 mice were daily exposed to either nebulized HQ or PBS for 1 h for a total of eight weeks. At weeks 6 and 8, the mice were primed and boosted with the trivalent influenza vaccine via IM respectively. Although the HQ exposure did not alter the body weight of the mice and the biochemical and hematological parameters, the pollutant increased the oxidative stress in splenocytes of immunized animals, modified the morphology of spleen follicles, and augmented the size of their lymph nodes. The lymphoid organs of HQ-exposed mice presented a similar number of vaccine-specific IgG-secreting cells, titers of vaccine-specific total IgG, and respective subclasses. Transcriptome studies with HQ, benzene, or cigarette smoke exposure were also analyzed. The genes up-regulated upon pollutant exposure were associated with neutrophil migration and were shown to be co-expressed with antibody-secreting cell genes. Therefore, these findings suggest that HQ exposure may trigger an immune-compensatory mechanism that enhances the humoral responses induced by influenza vaccination.

São Paulo School of Advanced Sciences on Vaccines: an overview

Two years ago, we held an exciting event entitled the São Paulo School of Advanced Sciences on Vaccines (SPSASV). Sixty-eight Ph.D. students, postdoctoral fellows and independent researchers from 37 different countries met at the Mendes Plaza Hotel located in the city of Santos, SP - Brazil to discuss the challenges and the new frontiers of vaccinology. The SPSASV provided a critical and comprehensive view of vaccine research from basics to the current state-of-the-art techniques performed worldwide. For 10 days, we discussed all the aspects of vaccine development in 36 lectures, 53 oral presentations and 2 poster sessions. At the end of the course, participants were further encouraged to present a model of a grant proposal related to vaccine development against individual pathogens. Among the targeted pathogens were viruses (Chikungunya, HIV, RSV, and Influenza), bacteria (Mycobacterium tuberculosis and Streptococcus pyogenes), parasites (Plasmodium falciparum or Plasmodium vivax), and the worm Strongyloides stercoralis. This report highlights some of the knowledge shared at the SPSASV.(AU)

Long noncoding RNAs are involved in multiple immunological pathways in response to vaccination.

Understanding the mechanisms of vaccine-elicited protection contributes to the development of new vaccines. The emerging field of systems vaccinology provides detailed information on host responses to vaccination and has been successfully applied to study the molecular mechanisms of several vaccines. Long noncoding RNAs (lncRNAs) are crucially involved in multiple biological processes, but their role in vaccine-induced immunity has not been explored. We performed an analysis of over 2,000 blood transcriptome samples from 17 vaccine cohorts to identify lncRNAs potentially involved with antibody responses to influenza and yellow fever vaccines. We have created an online database where all results from this analysis can be accessed easily. We found that lncRNAs participate in distinct immunological pathways related to vaccine-elicited responses. Among them, we showed that the expression of lncRNA FAM30A was high in B cells and correlates with the expression of immunoglobulin genes located in its genomic vicinity. We also identified altered expression of these lncRNAs in RNA-sequencing (RNA-seq) data from a cohort of children following immunization with intranasal live attenuated influenza vaccine, suggesting a common role across several diverse vaccines. Taken together, these findings provide evidence that lncRNAs have a significant impact on immune responses induced by vaccination.

Immune Priming and Long-term Persistence of Memory B Cells After Inactivated Poliovirus Vaccine in Macaque Models: Support for at least 2 Doses.

Background: As a risk-mitigation strategy to minimize paralytic polio following withdrawal of Sabin type 2 from the oral poliovirus vaccine in April 2016, a single full dose or 2 fractional doses of inactivated poliovirus vaccine (IPV) are recommended. However, limited knowledge exists on long-term persistence of immune memory following 1- or 2-dose IPV schedules. Methods: We examined induction and maintenance of immune memory following single- vs 2-dose IPV schedules, either full-dose intramuscular or fractional-dose intradermal, in rhesus macaques. Humoral responses, bone marrow-homing antibody-secreting plasma cells, and blood-circulating/lymph node-homing memory B cells were examined longitudinally. Results: A single dose of IPV, either full or fractional, induced binding antibodies and memory B cells in all vaccinated macaques, despite failing to induce neutralizing antibodies (NT Abs) in many of them. However, these memory B cells declined rapidly, reaching below detection in the systemic circulation by 5 months; although a low frequency of memory B cells was detectable in draining lymph nodes of some, but not all, animals. By contrast, a 2-dose vaccination schedule, either full or fractional, efficiently induced NT Abs in all animals along with bone marrow-homing plasma cells and memory B cells. These memory B cells persisted in the systemic circulation for up to 16 months, the maximum duration tested after the second dose of vaccination. Conclusions: Two doses of IPV, regardless of whether fractional or full, are more effective than a single dose for inducing long-lasting memory B cells.

To B or Not to B: Understanding B Cell Responses in the Development of Malaria Infection.

Malaria is a widespread disease caused mainly by the Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) protozoan parasites. Depending on the parasite responsible for the infection, high morbidity and mortality can be triggered. To escape the host immune responses, Plasmodium parasites disturb the functionality of B cell subsets among other cell types. However, some antibodies elicited during a malaria infection have the potential to block pathogen invasion and dissemination into the host. Thus, the question remains, why is protection not developed and maintained after the primary parasite exposure? In this review, we discuss different aspects of B cell responses against Plasmodium antigens during malaria infection. Since most studies have focused on the quantification of serum antibody titers, those B cell responses have not been fully characterized. However, to secrete antibodies, a complex cellular response is set up, including not only the activation and differentiation of B cells into antibody-secreting cells, but also the participation of other cell subsets in the germinal center reactions. Therefore, a better understanding of how B cell subsets are stimulated during malaria infection will provide essential insights toward the design of potent interventions.

Immune Cell Dynamics in Rhesus Macaques Infected with a Brazilian Strain of Zika Virus.

Zika virus (ZIKV) is a mosquito-borne and sexually transmitted flavivirus that is associated with fetal CNS-damaging malformations during pregnancy in humans. This study documents the viral kinetics and immune responses in rhesus macaques infected with a clinical ZIKV Brazilian isolate. We evaluated the viral kinetics and immune responses induced after an i.v. infection with a Brazilian ZIKV clinical isolate (HS-2015-BA-01) in rhesus macaques for up to 142 d. ZIKV-specific Ab-secreting cells, germinal center reactions, and monocyte, dendritic cell, NK, and T cell frequencies were monitored. ZIKV loads were readily detected in plasma (until day 5 or 7), semen and urine (until days 7 and 14), and saliva (until day 42), but the viremia was rapidly controlled. No detectable clinical manifestations were observed. However, lymph node hyperplasia was clearly visible postviremia but was associated with low frequencies of ZIKV-specific Ab-secreting cells in lymph nodes and bone marrow, correlating with low Ab titers. CD14+/CD16- monocytes and myeloid CD11chi dendritic cells decreased in blood, whereas NK and T cell numbers were only marginally altered during the course of the study. ZIKV infection caused a significant lymphoid tissue activation but limited induction of ZIKV-specific B cells, suggesting that these parameters need to be considered for ZIKV vaccine design.

Adjuvanting a Simian Immunodeficiency Virus Vaccine with Toll-Like Receptor Ligands Encapsulated in Nanoparticles Induces Persistent Antibody Responses and Enhanced Protection in TRIM5α Restrictive Macaques.

Our previous work has shown that antigens adjuvanted with ligands specific for Toll-like receptor 4 (TLR4) and TLR7/8 encapsulated in poly(lactic-co-glycolic) acid (PLGA)-based nanoparticles (NPs) induce robust and durable immune responses in mice and macaques. We investigated the efficacy of these NP adjuvants in inducing protective immunity against simian immunodeficiency virus (SIV). Rhesus macaques (RMs) were immunized with NPs containing TLR4 and TLR7/8 agonists mixed with soluble recombinant SIVmac239-derived envelope (Env) gp140 and Gag p55 (protein) or with virus-like particles (VLPs) containing SIVmac239 Env and Gag. NP-adjuvanted vaccines induced robust innate responses, antigen-specific antibody responses of a greater magnitude and persistence, and enhanced plasmablast responses compared to those achieved with alum-adjuvanted vaccines. NP-adjuvanted vaccines induced antigen-specific, long-lived plasma cells (LLPCs), which persisted in the bone marrow for several months after vaccination. NP-adjuvanted vaccines induced immune responses that were associated with enhanced protection against repeated low-dose, intravaginal challenges with heterologous SIVsmE660 in animals that carried TRIM5α restrictive alleles. The protection induced by immunization with protein-NP correlated with the prechallenge titers of Env-specific IgG antibodies in serum and vaginal secretions. However, no such correlate was apparent for immunization with VLP-NP or alum as the adjuvant. Transcriptional profiling of peripheral blood mononuclear cells isolated within the first few hours to days after primary vaccination revealed that NP-adjuvanted vaccines induced a molecular signature similar to that induced by the live attenuated yellow fever viral vaccine. This systems approach identified early blood transcriptional signatures that correlate with Env-specific antibody responses in vaginal secretions and protection against infection. These results demonstrate the adjuvanticity of the NP adjuvant in inducing persistent and protective antibody responses against SIV in RMs with implications for the design of vaccines against human immunodeficiency virus (HIV). IMPORTANCE: The results of the RV144 HIV vaccine trial, which demonstrated a rapid waning of protective immunity with time, have underscored the need to develop strategies to enhance the durability of protective immune responses. Our recent work in mice has highlighted the capacity of nanoparticle-encapsulated TLR ligands (NP) to induce potent and durable antibody responses that last a lifetime in mice. In the present study, we evaluated the ability of these NP adjuvants to promote robust and durable protective immune responses against SIV in nonhuman primates. Our results demonstrate that immunization of rhesus macaques with NP adjuvants mixed with soluble SIV Env or a virus-like particle form of Env (VLP) induces potent and durable Env-specific antibody responses in the serum and in vaginal secretions. These responses were superior to those induced by alum adjuvant, and they resulted in enhanced protection against a low-dose intravaginal challenge with a heterologous strain of SIV in animals with TRIM5a restrictive alleles. These results highlight the potential for such NP TLR L adjuvants in promoting robust and durable antibody responses against HIV in the next generation of HIV immunogens currently being developed.

Vaccine-induced plasmablast responses in rhesus macaques: phenotypic characterization and a source for generating antigen-specific monoclonal antibodies.

Over 100 broadly neutralizing antibodies have been isolated from a minority of HIV infected patients, but the steps leading to the selection of plasma cells producing such antibodies remain incompletely understood, hampering the development of vaccines able to elicit them. Rhesus macaques have become a preferred animal model system used to study SIV/HIV, for the characterization and development of novel therapeutics and vaccines as well as to understand pathogenesis. However, most of our knowledge about the dynamics of antibody responses is limited to the analysis of serum antibodies or monoclonal antibodies generated from memory B cells. In a vaccine setting, relatively little is known about the early cellular responses that elicit long-lived plasma cells and memory B cells and the tools to dissect plasmablast responses are not available in macaques. In the current study, we show that the majority (>80%) of the vaccine-induced plasmablast response are antigen-specific by functional ELISPOT assays. While plasmablasts are easily defined and isolated in humans, those same phenotypic markers have not been useful for identifying macaque plasmablasts. Here we describe an approach that allows for the isolation and single cell sorting of vaccine-induced plasmablasts. Finally, we show that isolated plasmablasts can be used to efficiently recover antigen-specific monoclonal antibodies through single cell expression cloning. This will allow detailed studies of the early plasmablast responses in rhesus macaques, enabling the characterization of both their repertoire breadth as well as the epitope specificity and functional qualities of the antibodies they produce, not only in the context of SIV/HIV vaccines but for many other pathogens/vaccines as well.

Therapeutic vaccination against the rhesus lymphocryptovirus EBNA-1 homologue, rhEBNA-1, elicits T cell responses to novel epitopes in rhesus macaques.

Epstein-Barr virus (EBV) is a vaccine/immunotherapy target due to its association with several human malignancies. EBNA-1 is an EBV protein consistently expressed in all EBV-associated cancers. Herein, EBNA-1-specific T cell epitopes were evaluated after AdC-rhEBNA-1 immunizations in chronically lymphocryptovirus-infected rhesus macaques, an EBV infection model. Preexisting rhEBNA-1-specific responses were augmented in 4/12 animals, and new epitopes were recognized in 5/12 animals after vaccinations. This study demonstrated that EBNA-1-specific T cells can be expanded by vaccination.